Francisella tularensis is a Category A select agent that can cause severe and fatal disease in humans if an aerosol containing as few as 10-100 CFU is inhaled. It is a category A select agent and is a priority for countermeasure development. The ability of the Type B derived live vaccine strain, LVS, to confer partial protection against aerosol Type A challenge in men provided proof of principle that a live attenuated F. tularensis vaccine could offer partial protection. However, LVS is not licensed by the FDA and suffers from several drawbacks that render it a sub-optimal tularemia vaccine. An optimal live vaccine will be based on introducing precisely defined attenuating mutations into a Type A parent strain and will confer strong protection against exposure to aerosolized virulent Type A F. tularensis. We have developed genetic tools that facilitated engineering of specifically targeted mutations in Type A F. tularensis. Six of the candidate strains from this series tested thus far, demonstrated attenuation in the mouse and/or macrophage survival assays. One of our most advanced candidates, FTT1103, has been shown to confer protection against wild type, Type A challenge in the stringent C57BL/6 mouse model. No other vaccine published in the literature has shown this protective capacity. In this grant application we will make use of the data produced and expertise acquired from the initial Mid Atlantic RCE funding investment to advance promising vaccine candidates to preclinical studies that will confirm their immunogenic and protective potential. Parallel studies using the rabbit and mouse models will allow accelerated progress towards the identification of lead vaccine candidates. These studies will validate the use of these two animal species as relevant models for evaluation of live Francisella vaccine strains, information that is not yet verified. In addition, in vitro assessment of vaccine strain interaction and cytokine induction in infected macrophages will be performed. Our previous studies demonstrated that mutations in Type A strains had significant effects on these critical processes and we hypothesize that a correlation could be made between macrophage data and animal model immunogenicity/efficacy that could suggest a correlate of protection or at the least, essential in vitro responses. Finally, the vaccine candidate that demonstrates protective capacity in the rabbit model will undergo expanded safety studies investigating dose ranges that will be important for advancement. This concerted effort by the assembled experts will allow identification of a promising live attenuated Francisella vaccine strain.